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w Patterning in Development > 6. Limb patterning > Flashcards

6. Limb patterning Flashcards

1
Q

Segment polarity and homeotic genes

A
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2
Q

What could be the genetic manipulations in limb development to understand patterning?

A

Modifications to observe gene expression:
- gene KI
- gene KO
- cDNA overexpression
- genomic BAC constructs

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3
Q

Explain conditional gene targeting

A

Introduction of Cre loxP system - when Cre added - gene turned off/on

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4
Q

Explain the structure of a limb bud

A
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5
Q

What are the limb axes that need to be patterned

A
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6
Q

What are the initial signals in limb bud induction?

A

Limb induction in lateral plate mesoderm (LPM):
- Fgf10 expressed
- Wnt8c stabilized in hindlimb
- Wnt2b stabilized in forelimb
- in the set limb bud regions Fgf10 -> Fgf8 expression => apical ectodermal ridge (AER) formation

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7
Q

What is the function of Fgf10 in limb development?

A

Fgf10 necessary for for limb bud induction => Fgf10 KO - no limbs - some initial outgrowth but no limb bud outgrowth

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8
Q

What if exogenous Fgf is introduced in Fgf10-/-?

A

Ectopic Fgf beads induce limb formation - type of limb depends on Tbx4/5

Tbx5 - forelimb
Tbx4 - hindlimb
-> but later found that not these are the determinants of fore vs hind limb diffferentiation - but actually retinoic acid -> Hox genes control fore vs hind

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9
Q

How was Tbx role in fore vs hind limb development disproved?

A

Thought that Tbx5 / Tbx4 - but in Tbx5-/- + ectopic Tbx4 -> still developed as forelimb => Tbx4 replaced Tbx5

Both Tbx play similar role - what actually controls is site restricted => retinoic acid -> Hox genes

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10
Q

What controls fore vs hind limb development in limb buds?

A

Retinoic acid (RA) signaling regionalizes lateral plate mesoderm (LPM) from which limb buds outgrow - RA regionalizes LPM by regulating Hox expression =>
- rostral Hox (fore)
- caudal Hox (hind)

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11
Q

What is the sequence of signalling in fore vs hind limb induction?

A
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12
Q

What is the main determining region for P-D axis development in the limb?

A

Apical ectodermal ridge (AER) - necessary for outgrowth of limb bud - AER responsible for P-D patterning - cells under AER = progress zone (PZ)

Limb outgrowth develops sequentially - progress established as time develops - the later AER removed - the more distal development established

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13
Q

Explain limb bud anatomy

A

Limb bud anatomy:
- apical ectodermal ridge (AER): P-D pattern
- progress zone (PZ): limb outgrowth
- zone of polarising activity (ZPA): A-P pattern

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14
Q

Explain progress zone in the limb bud

A

Progress zone (PZ) can only divide and contribute to outgrowth while in contact with AER - the more time spent - the more distal structures developed

When lose contact with AER - differentiates into cartilage

Experiment showed that removal of AER -> cell death + decreased proliferation => progenitor cells outside AER can induce outgrowth but not sufficiently enough

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15
Q

What is the most important signalling molecule in P-D axis development in the limb?

A

In P-D development FGFs important:
(most proximal) Fgf8 > Fgf4 > Fgf9 > Fgf17 (most distal) in terms of importance

Fgf8 -> activates RA - RA diffuses P-D -> activates Meis/Meis2 proximal gene + Hoxa13, Hoxa11 distal genes

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16
Q

What is the role of Meis in P-D axis in limb development?

A

Meis is a proximal gene:
1. Meis expressed homogenously through P-D axis
2. FGF expressed in AER -> inhibits Meis expression in distal => Meis gradient in P-D axis
3. Meis inhibited -> HoxA cluster - Hoxa11, Hoxa13

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17
Q

Explain how limb A-P axis is patterned?

A

A-P axis of the limb patterned from the posterior - zone of polarising activity (ZPA) - signals - morphogen conc gradient - Shh

Ecotpic RA beads also induce axis duplication as ZPA grafting

18
Q

Explain hte importance of Shh in all of development

A

Shh is a major regulator of development - acts as a morphogen - acts many times in development for different functions - Shh-/- mutants very defected - no distal limb, no head, no normal eye development,

19
Q

Explain hedgehog -> sonic hedgehog gene relation

A

Hedgehog gene - Drosophila gene - called hedgehog because -/- mutants have shortened trunk + all covered in dentacles - instead of forming segments

Sonic hedgehog gene - homolg in vertebrates

20
Q

How Shh expression was mapped?

A

Shh was fate mapped - Shh expressing cells adopt more posterior fate - not contributing to digits 1 and 2

21
Q

Explain biphasic model

22
Q

What is the role of Gli gene and how does it relate to limb patterning?

A

Gli acts in A-P patterning of the limb - effector of Shh - secreted in posterior - diffuses into anterior more than Shh - Shh and Gli3 are dispensable for limb skeleton formation - regulate digit number and identity

Gli3 downstream of Shh, mjust oth be ative for effect - if Shh-/- -> limbs can still develop some with only Gli3 - but if double KO Gli3-/-, Shh-/- -> same phenotype as Gli3-/-

23
Q

Explain ZRS related enhancer in Shh expression

A

ZRS (ZPA regulatory sequence) enhancer required for Shh expression in the limb - mutations result in ZRS associated syndromes

24
Q

Why is it hard to change Hox gene expression experimentally?

A

Hox genes difficult to investigate because arranged in a cluster - in a specific architecture - **if smth changed **- disrupts whole expression regulation - which comes externally -> Hox cluster architecture needs to be intact to be appropriatly regulated

25
Explain the feedback loop in gene expression of Hox and Shh in limb development
26
What is the first gene expressed in cartilage formation?
Sox9
27
Explain what is a turing type reaction diffusion model
**Turing type reaction diffusion model** - two molecules acts as an **activator and inhibitor** - both **diffuse** in an area - **activator** activates **production** of **inhibitor + itself **- **inhibitor** **inhibits** **activator** => over time **oscillations** of expressoion
28
Where is the Turing Network used in limb patterning
**Turing network model** used by **Hox** genes to **generate periodicity in digit** formation
29
Explain how Shh, Fgf, Gli3R, Wnt, Sox9, BMP and Hox genes are related in limb development
30
What are the important factors in DV patterning of the limb?
D-V patterning **determined by ectodermal component**: **BMP** signalling via **BMPR1a -> En1** in ventral ectoderm -> **restricts Wnt7** in dorsal ectoderm -> **Wnt7 induces Lmx1b** **Lmx1b** responsible for **dorsal** limb identity - Lmx1b -/- nail patella syndrome
31
Explain evolutionary tweaking in limb development
Between species **genes the same** but their **expression** is **altered** - specific to the species
32
Give an example how different species express same genes but differently to develop limbs
**Evolutionary conserved genes** but **expressed differently** between species - ex: **Shh expression in ZPA** in limb development of **bats** is **delayed** and **expanded** early - later **expression reinitiated** for **inter-digital tissue**
33
How mice were engineered to express snake limb developmental programme
**Proved** that **evolutionary conserved** mechanisms of **limb development** between different species
34
How does Shh expression differ in animals with fewer digits?
In **fewer digit** animals **Shh turned off earlier** - however - **not in cows**: Shh expression **early** like in mouse - also **expansion of Shh** protein and signalling **but not Otch1**
35
Explain Shh signalling outcome in limb development
36
How do limb development genes act in regeneration
**Salamanders** regenerate limbs: - **RA proximalizes regeneration** - **muscle** does not respect - **no P-D** boundries - **connective tissue** **responds** to **P-D** boundries - remember their expression levels of Meis => in **regeneration** **blastema** cells have **P-D determination system** from connective tissues
37
smth blastema patterning in regeneration
38
What is the similarity between vertebrate limb patterning and Drosophila?
Vertebrate limb bud <=> Drosophila wing imaginal disc
39
Relationship between ZPA, Shh, AER, FGF, polarization and proliferation in limb development
40
check paper discussion notes for more info

w Patterning in Development (12 decks)

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